An osteoclast is unique in being exposed locally to high millimolar Ca2+ levels resulting from mineral dissolution. We recently discovered that high ambient Ca2+ activates a ryanodine receptor-gated Ca2+ channel located, quite unusually, in the cell's plasma membrane. Classically, however, ryanodine receptors function as Ca2+ release channels at their microsomal, and more recently discovered, nuclear membrane locations. They are gated primarily by Ca2+ and the second messenger, cyclic adenosine diphosphate-ribose (cADPr). The latter is formed from NAD+ following its cyclization by the enzyme, CD38 (an ADP-ribosyl cyclase). We provide compelling preliminary data demonstrating that: (a) CD38 mRNA is expressed in the osteoclast; (b) immunoreactive CD38 is localized to the cell's plasma membrane; (c) when activated, CD38 triggers a cytosolic Ca2+ signal likely via cADPr generation from NAD+; and (d) CD38-induced Ca2+ signaling is associated with resorption inhibition and enhanced interleukin-6 secretion. Our goal is to examine whether CD38, by converting NAD+ to cADPr, regulates osteoclast Ca2+ homeostasis and hence, bone resorption and cytokine gene expression. Specifically, we will first examine whether cADPr, generated from NAD+ through CD38 catalysis, triggers cytosolic and nucleoplasmic Ca2+ transients via ryanodine receptor activation at the plasma, microsomal, and nuclear membranes. For this, we will use 'functional' CD38 antibodies and cADPr inhibitors together with state-of-the-art single cell and nuclear Ca2+ microfluorimetry, patch clamp electrophysiology, and VOXEL-assisted confocal microscopy. Next, using the pit (resorption) assay, together with in situ RT-PCR cytoimaging and the RNase protection assay, we propose to investigate the mechanism through which CD38 inhibits bone resorption, but paradoxically enhances interelukin-6 expression. Finally, we shall study any possible feedback regulation of CD38 gene expression by interleukin-6 and Ca2+ again utilizing in situ RT-PCR cytoimaging and RNase protection assays. To determine whether effects are transcriptional, and having cloned the full-length CD38 cDNA, we shall soon be poised to measure activity of the CD38 gene promoter following its cloning and characterization from a rabbit genomic library. Taken together, the studies should provide mechanistic insights into the role of the NAD+/CD38/cADPr/Ca2+ system in osteoclast control.